This invention relates to plant cellulose synthase cDNA encoding sequences, and their use in modifying plant phenotypes. Methods are provided whereby the sequences can be used to control or limit the expression of endogenous cellulose synthase.
This invention also relates to methods of using in vitro constructed DNA transcription or expression, cassettes capable of directing fiber-tissue transcription of a DNA sequence of interest in plants to produce fiber cells having an altered phenotype, and to methods of providing for or modifying various characteristics of cotton fiber. The invention is exemplified by methods of using cotton fiber promoters for altering the phenotype of cotton fiber, and cotton fibers produced by the method.
In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of the major cell wall polymer of plants, cellulose.
Numerous efforts have been directed toward the study of synthesis of cellulose (1,4-xcex2-D-glucan) in higher plants. However, hampered by low rates of activity in vitro, the cellulose synthase of plants has resisted purification and detailed characterization (for reviews, see 1,2). Aided by the discovery of cyclic-di-GMP as a specific activator, the cellulose synthase of the bacterium Acetobacter xylinum can be easily assayed in vitro, has been purified to homogeneity, and a catalytic subunit identified (for reviews, see 2,3). Furthermore, an operon of four genes involved in cellulose synthesis in A. xylinum has been cloned (4-7).
Characterization of these genes indicates that the first gene, termed either BcsA (7) or AcsAB (6) codes for the 83 kD subunit of the cellulose synthase that binds the substrate UDP-glc and presumably catalyzes the polymerization of glucose residues to 1,4-xcex2-D-glucan (8). The second gene (B) of the operon is believed to function as a regulatory subunit binding cyclic-di-GMP (9) while recent evidence suggests that the C and D genes may code for proteins that form a pore allowing secretion of the polymer and control the pattern of crystallization of the resulting microfibrils (6).
Recent studies with another gram-negative bacterium, Agrobacterium tumefaciens, have also led to cloning of genes involved in cellulose synthesis (10,11), although the proposed pathway of synthesis differs in some respects from that of A. xylinum. In A. tumefaciens, a CelA gene showing significant homology to the BcsA/AcsAB gene of A. xylinum, is proposed to transfer glc from UDP-glc to a lipid acceptor; other gene products may then build up a lipid oligosaccharide that is finally polymerized to cellulose by the action of an endo-glucanase functioning in a synthetic mode. In addition, homologs of the CelA, B, and C genes have been identified in E. coli, but, as this organism is not known to synthesize cellulose in vivo, the function of these genes is not clear (2).
These successes in bacterial systems opened the possibility that homologs of the bacterial genes might be identified in higher plants. However, experments in a number of laboratories utilizing the A. xylinum genes as probes for screening plant cDNA libraries have failed to identify similar plant genes. Such lack of success suggests that, if plants do contain homologs of the bacterial genes, their overall sequence homology is not very high. Recent studies analyzing the conserved motifs common to glycosyltransferases using either UDP-glc or UDP-GlcNAc as substrate suggest that there are specific conserved regions that might be expected to be found in any plant homolog of the catalytic subunit (referred to hereafter as CelA). In one of these studies, Delmer and Amor (2) identifed a motif common to many such glycosyltransferases including the bacterial CelA proteins. An independent analysis (6) also concluded that this motif was highly conserved in a group of similar glycosyltransferases.
Extending these studies further, Saxena et al. (12) presented an elegant model for the mechanism of catalysis for enzymes such as cellulose synthase that have the unique problem of synthesizing consecutive residues that are rotated approximately rotated 180xc2x0 with respect to each other. The model invokes independent UDP-glc binding sites and, based upon hydrophobic cluster analysis of these enzymes, the authors concluded that 3 critical regions in all such processive glycosyltransferases each contain a conserved aspartate (D) residue, while a fourth region contained a conserved QXXRW motif. The first D residue resides in the motif as previously analyzed (2,6).
In general, genetic engineering techniques have been directed to modifying the phenotype of individual prokaryotic and eukaryotic cells, especially in culture. Plant cells have proven more intransigent than other eukaryotic cells, due not only to a lack of suitable vector systems but also as a result of the different goals involved. For many applications, it is desirable to be able to control gene expression at a particular stage in the growth of a plant or in a particular plant part. For this purpose, regulatory sequences are required which afford the desired initiation of transcription in the appropriate cell types and/or at the appropriate time in the plant""s development without having serious detrimental effects on plant development and productivity. It is therefore of interest to be able to isolate sequences which can be used to provide the desired regulation of transcription in a plant cell during the growing cycle of the host plant.
One aspect of this interest is the ability to change the phenotype of particular cell types, such as differentiated epidermal cells that originate in fiber tissue, i.e. cotton fiber cells, so as to provide for altered or improved aspects of the mature cell type. Cotton is a plant of great commercial significance. In addition to the use of cotton fiber in the production of textiles, other uses of cotton include food preparation with cotton seed oil and animal feed derived from cotton seed husks.
A related goal involving the control of cell wall and characteristics would be to affect valuable, secondary tree characteristics of wood for paper forestry products. For instance, by altering the balance of cellulose and lignin, the quality of wood for paper production may be improved.
Finally, despite the importance of cotton as a crop, the breeding and genetic engineering of cotton fiber phenotypes has taken place at a relatively slow rate because of the absence of reliable promoters for use in selectively effecting changes in the phenotype of the fiber. In order to effect the desired phenotypic changes, transcription initiation regions capable of initiating transcription in fiber cells during development are desired. Thus, an important goal of cotton bioengineering research is the acquisition of a reliable promoter which would permit expression of a protein selectively in cotton fiber to affect such qualities as fiber strength, length, color and dyability.
Relevant Literature
Cotton fiber-specific promoters are discussed in PCT publications WO 94/12014 and WO 95/08914, and John and Crow, Proc. Natl. Acad. Sci. USA, 89:5769-5773, 1992. cDNA clones that are preferentially expressed in cotton fiber have been isolated. One of the clones isolated corresponds to mRNA and protein that are highest during the late primary cell wall and early secondary cell wall synthesis stages. John and Crow, supra.
In plants, control of cytoskeletal organization is poorly understood in spite of its importance for the regulation of patterns of cell division, expansion, and subsequent deposition of secondary cell wall polymers. The cotton fiber represents an excellent system for studying cytoskeletal organization. Cotton fibers are single cells in which cell elongation and secondary wall deposition can be studied as distinct events. These fibers develop synchronously within the boll following anthesis, and each fiber cell elongates for about 3 weeks, depositing a thin primary wall (Meinert and Delmer, (1984) Plant Physiol. 59: 1088-1097; Basra and Malik, (1984) Int Rev of Cytol 89: 65-113). At the time of transition to secondary wall cellulose synthesis, the fiber cells undergo a synchronous shift in the pattern of cortical microtubule and cell wall microfibril alignments, events which may be regulated upstream by the organization of actin (Seagull, (1990) Protoplasma 159: 44-59; and (1992) In: Proceedings of the Cotton Fiber Cellulose Conference, National Cotton Council of America, Memphis RN, pp 171-192.
Agrobacterium-mediated cotton transformation is described in Umbeck, U.S. Pat. Nos. 5,004,8631 and 5,159,135 and cotton transformation by particle bombardment is reported in WO 92/15675, published Sep. 17, 1992. Transformation of Brassica has been described by Radke et al. (Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992) 11:499-505.
Genes involved in lignin biosynthesis are described by Dwivedi, U. N., Campbell, W. H., Yu, J., Datla., R. S. S., Chiang, V. L., and Podila, G. K. (1994) xe2x80x9cModification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populusxe2x80x9d Pl. Mol. Biol. 26: 61-71; and Tsai, C. J., Podila, G. K. and Chaing, V. L. (1995) xe2x80x9cNucleotide sequence of Populus tremuloides gene for caffeic acid/5 hydroxyferulic acid O-methyltransferasexe2x80x9d Pl. Physiol. 107: 1459; and also U.S. Pat. No. 5,451,514 (claiming the use of cinnamyl alcohol dehydrogenase gene in an antisense orientation such that the endogenous plant cinnamyl alcohol dehydrogenase gene is inhibited).
1. Gibeaut, D. M., and Carpita, N. C. (1994) FASEB J. 8, 904-915.
2. Delmer, D. P., and Amor, Y. (1995) Plant cell 7, 987-1000.
3. Ross, P., Mayer, R., and Benziman, M. (1991) Microbiol. Rev. 55, 35-58.
4. Saxena, I. M., Lin, F. C., and Brown, R. M., Jr. (1990) Plant Mol. Biol. 15, 673-683.
5. Saxena, I. M., Lin, F. C., and Brown, R. M., Jr. (1992) Plant Mol. Biol. 16, 947-954.
6. Saxena, I. M., Kudlicka, K., Okuda, K., and Brown, R. M., Jr. (1994) J. Bacteriol. 176, 5735-5752.
7. Wong, H. C., Fear, A. L., Calhoon, R. D., Eidhinger, G. H., Mayer, R., Amikam, D., Benziman, M., Gelfand, D. H., Meade, J. H., Emerick, A. W., Bruner, R., Ben-Basat, B. A., and Tal, R. (1990) Proc. Natl. Acad. Sci. USA 87, 8130-8134.
8. Lin, F.-C., Brown, R. M. Jr., Drake, R. R. Jr., and Haley, B. E. (1990) J. Biol. Chem. 265, 4782-4784.
9. Mayer, R., Ross, P., Winhouse, H., Amikm, D., Volman, G., Ohana, P., Calhoon, R. D., Wong, H. C., Emerick, A. W., and Benziman, M. (1991) Proc. Natl. Acad. Sci. USA 88, 5472-5476.
10. Matthysse, A. G., White, S., and Lightfoot, R. (1995a) J. Bacteriol. 177, 1069-1075.
11. Matthysse, A. G., Thomas, D. O. L., and White, S. (1995b) J. Bacteriol. 177, 1076-1081.
12. Saxena, I. M., Brown, R. M.,Jr., Fevre, M., Geremia, R. A., and Henrissat, B. (1995) J. Bacteriol. 177, 1419-1424.
13. Meinert, M., and Delmer, D. P. (1977) Plant Physiol. 59, 1088-1097.
14. Delmer, D. P., Pear, J. R., Andrawis, A., and Stalker, D. M. (1995) Mol. Gen. Genet. 248, 43-51.
15. Delmer, D. P., Solomon, M., and Read, S. M. (1991) Plant Physiol. 95, 556-563.
16. Nagai, K., and Thogersen, H. C. (1987) Methods in Enzymol. 153, 461-481.
17. Laemmli, U. K. (1970) Nature 227, 680-685.
18. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132.
19. Oikonomakos, N. G., Acharya, K. R., Stuart, D. I., Melpidou, A. E., McLaughlin, P. J., and Johnson, L. N. (1988) Eur. J. Biochem. 173, 569-578.
20. Maltby, D., Carpita, N. C., Montezinos, D., Kulow, C., and Delmer, D. P. (1979) Plant Physiol. 63, 1158-1164.
21. Inoue, S. B., Takewaki, N., Takasuka, T., Mio, T., Adachi, M., Fujii, Y., Miyamoto, C., Arisawa, M., Furuichi, Y., and Watanabe, T. (1995) Eur. J. Biochem. 231, 845-854.
22. Jacob, S. R., and Northcote, D. H. (1985) J. Cell Sci. 2 (suppl.), 1-11.
23. Delmer, D. P. (1987) Annu. Rev. Plant Physiol. 38, 259-290.
24. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410
25. Milligan, G., Parenti, M., and Magee, A. I. (1995) TIBS 20, 183-186.
26. Amor, Y., Haigler, C. H., Johnson, S., Wainscott, M., and Delmer, D. P. (1995) Proc. Natl. Acad. Sci. USA 92, 9353-9357.
27. Amor, Y., Mayer, R., Benziman, M., and Delmer, D. P. (1991) Plant Cell 3, 989-995.
Two cotton genes, CelA1 and CelA2, have been shown to be highly expressed in developing fibers at the onset of secondary wall cellulose synthesis. Comparisons indicate that these genes and the rice CelA gene encode polypeptides that have three regions of reasonably high homology, both in terms of primary amino acid sequence and hydropathy, with bacterial CelA proteins. The fact that these homologous stretches are in the same sequential order as in the bacterial CelA proteins and also contain four sub-regions previously predicted to be critical for substrate binding and catalysis (12) argues that the plant genes encode true homologs of bacterial CelA proteins. Furthermore, the pattern of expression in fiber as well as our demonstration that at least one of these highly-conserved regions is critical for UDP-glc binding also supports this conclusion.
Novel DNA promoter sequences are also supplied, and methods for their use are described for directing transcription of a gene of interest in cotton fiber.
The developing cotton fiber is an excellent system for studies on cellulose synthesis as these single cells develop synchronously in the boll and, at the end of elongation, initiate the synthesis of a nearly pure cellulosic cell wall. During this transition period, synthesis of other cell wall polymers ceases and the rate of cellulose synthesis is estimated to rise nearly 100-fold in vivo (13). In our continuing efforts to identify genes critical to this phase of fiber development, we have initiated a program sequencing randomly selected cDNA clones derived from a library prepared from mRNA harvested from fibers at the stage in which secondary wall synthesis approaches its maximum rate (approximately 21 dpa).
We have characterized two cotton (Gossypium hirsutum) cDNA clones and identified one rice (Oryza sativa) cDNA that are homologs of the bacterial CelA genes that encode the catalytic subunit of cellulose synthase. Three regions in the deduced amino acid sequences of the plant CelA gene products are conserved with respect to the proteins encoded by bacterial CelA genes. Within these conserved regions are four highly conserved subdomains previously suggested to be critical for catalysis and/or binding of the substrate UDP-glc. An overexpressed DNA segment of the cotton CelA1 gene encodes a polypeptide fragment that spans these domains and effectively binds UDP-glc, while a similar fragment having one of these domains deleted does not. The plant CelA genes show little homology at the amino and carboxy terminal regions and also contain two internal insertions of sequence, one conserved and one hypervariable, that are not found in the bacterial gene sequences. Cotton CelA1 and CelA2 genes are expressed at high levels during active secondary wall cellulose synthesis in the developing fiber. Genomic Southern analyses in cotton demonstrate that CelA comprises a family of approximately four distinct genes.
We report here the discovery of two cotton genes that show highly-enhanced expression at the time of onset of secondary wall synthesis in the fiber. The sequences of these two cDNA clones, termed CelA1 and CelA2, while not identical are highly homologous to each other and to a sequenced rice EST clone discovered in the dBEST databank. The deduced proteins also share signifigant regions of homology with the bacterial CelA proteins. Coupled with their high level and specificity of expression in fiber at the time of active cellulose synthesis, as well as the ability of an E. coli expressed fragment of the CelA1 gene product to bind UDP-glc, these findings support the conclusion that these plant genes are true homologs of the bacterial CelA genes.
The methods of the present invention include transfecting a host plant cell of interest with a transcription or expression cassette comprising a cotton fiber promoter and generating a plant which is grown to produce fiber having the desired phenotype. Constructs and methods of the subject invention thus find use in modulation of endogenous fiber products, as well as production of exogenous products and in modifying the phenotype of fiber and fiber products. The constructs also find use as molecular probes. In particular, constructs and methods for use in gene expression in cotton embryo tissues are considered herein. By these methods, novel cotton plants and cotton plant parts, such as modified cotton fibers, may be obtained.
The sequences and constructs of this invention may also be used to isolate related cellulose synthase genes from forest tree species, for use in transforming and modifying wood quality. As and example, lignin, an undesirable by-product of the pulping process, by be reduced by over-expressing the cellulose synthase product and diverting production into cellulose.
Thus, the application provides constructs and methods of use relating to modification of cell and cell wall phenotype in cotton fiber and wood products.